The present invention relates to extended analogues of the mammalian hormone referred to as luteinising hormone-releasing hormone (LHRH). In particular, it relates to analogues of LHRH formed by adding to the C-terminus of the native LHRH amino acid sequence a short additional amino acid sequence ending with a cysteine residue, such that the conformation in solution is substantially unchanged, and polypeptide conjugates thereof suitable for raising anti-LHRH antibodies.
LHRH is a decapeptide having the amino acid sequence
pGlg-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH.sub.2 PA0 pGlg-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-Y-Z
wherein pglg represents a pyroglutamate residue and GlyNH.sub.2 represents glycine-amide, In mammals, this hormone is secreted by the hypothalamus and is responsible for controlling the release of luteinising hormone and follicle-stimulating hormone from the pituitary (Schally et al., Science (1973) 179, 341-350; Guillemin, R., Contraception (1972) 5, 1-32). The latter hormones are directly involved in controlling the development and the normal physiological functioning of testes and ovaries. PA1 wherein X represents Gly or a D-amino acid, Y represents one or more amino acid residues, which may be the same or different, and Z represents Cys or Tyr, such that the solution conformation of said analogue is substantially similar to that of native LHRH.
Active immunisation of various mammals to LHRH, eg. rats, rabbits, sheep and cattle, has been shown to lead in the case of males to testicular regression, reduction of testosterone secretion and cessation of spermatogensis and in the case of females to loss of cycling and ovarian regression, and it has long been appreciated that active immunisation to LHRH could provide a desirable means for routinely reducing fertility in domestic mammals and farm animals, particularly bull calves. In a comparative study of steer calves and immunologically-castrated calves produced by active immunisation to LHRH, the so-called "immunocastrates" were found to be superior in terms of their carcase composition since in these animals anabolic influence of the testes was not totally abolished (Robertson et al., Veterinary Record (1982) 111, 529-531). Moreover, surgical castration involves a degree of risk of infection and trauma, particularly if attempted on other than very young animals. Nevertheless, active immunisation to LHRH using a polypeptide conjugate of LHRH has previously required deleterious adjuvants and moreover the polypeptide conjugates themselves have been immunogenically unsatisfactory.
Hitherto published procedures for the conjugation of LHRH to a polypeptide carrier, e.g. bovine or human serum albumin, tetanus toxoid or thyroglobulin, have generally involved condensation of the hormone with a water-soluble carbodiimide. The absence of free carboxyl or amino moieties in LHRH means that coupling in such methods is probably effected through the hydroxyl group of Ser.sup.4 or via a carboxy-methylated derivative of His.sup.2. As a result, the immunogen is poorly-defined and unlikely to retain all the structural features of free LHRH in solution as considered desirable from the point of view of obtaining anti-LHRH antibodies capable of blocking functioning of LHRH in vivo; there is a danger that the peptide is attached through a region important for immunological recognition. Effective immunisation of mammals using such conjugates to provide a high titre of anti-LHRH antibodies capable of significantly reducing the biological efficacy of endogenous LHRH has only been achieved in the presence of an adjuvant liable to cause undesirable side effects, most commonly Freund's complete or incomplete adjuvant. Freund's complete adjuvant interferes with the tuberculin test in cattle and in addition this adjuvant and also Freund's incomplete adjuvant cause a variable amount of chronic inflammatory reaction at the site of injection. Such procedures have accordingly achieved no practical importance.
A totally synthetic LHRH vaccine based on muramyl dipeptide (MDP) has recently been shown to be capable of effecting immunological castration in male mice (Carelli et al., Proc. Nat. Acad. Sci. USA (1982) 79, 5392-5395; Carelli et al., Int. J. Immunopharmacol. (1985) 7, 215-224). However, due to the apparent pyrogenic effects of MDP, this technique of immunological castration is also likely to prove unacceptable for general use in veterinary practice.